[Changes in etoposide-induced apoptosis of HeLa tumor cells transfected with antisense oligonucleotide for BCL-2 mRNA].
Overexpression of genes involved in proliferation, including genes of BCL family, is often found in cells of most malignant tumors. Currently developed strategies of cancer treatment include trials combining classical chemotherapy with silencing of genes expression using the gene therapy. The aim of the study was to induce silencing of BCL-2 gene expression in HeLa tumor cell line using antisense oligonucleotides (ASOs) technique.


METHODS
Studies were carried out on in vitro HeLa cell cultures treated with etoposide. Cells were transfected by lipofection with ASO targeting BCL-2 mRNA. Effects of BCL-2 silencing were determined by Real-Time RT-PCR, proliferation/cytotoxicity MTT [3-(4,5-dimethylthiazol-2-yl)-2-5-dipheryl tetrazolium bromide] test, and by microscopic detection of apoptotic cells.


RESULTS
We showed a decrease in BCL-2 mRNA level in cells transfected with anti-BCL-2 ASO at concentration ranging from 50 to 1200 nM. Apoptotic cells were detected more frequently in transfected cultures compared with untreated controls. However, MTT tests did not display significant decrease of cell proliferation in the transfected cultures as compared with cultures treated with etoposide alone.


CONCLUSIONS
1. These results indicate that the studied ASO sequence-specifically decreases BCL-2 expression in HeLa cells in vitro, although its action is limited mostly by transfection efficiency. 2. The use of the anti-BCL-2 oligonucleotide in combination with etoposide results in significant deacrease of proliferation in cell cultures and this phenomenon is a result of synergy between used chemotherapy and gene therapy.
Bednarek
Ilona
I
Zakładu Biotechnologii i Inzynierii Genetycznej Slaskiego Uniwersytetu Medycznego w Katowicach. dribednarek@sum.edu.pl
Sypniewski
Daniel
D
Solarz
Joanna
J
Machnik
Grzegorz
G
Gałka
Sabina
S
Loch
Tomasz
T
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000235:genetics*; D049109:Cell Proliferation / Q000187:drug effects; D005047:Etoposide / Q000494:pharmacology*; D020868:Gene Silencing; D019254:Genes, bcl-2 / Q000187:drug effects*; D015316:Genetic Therapy; D006367:HeLa Cells / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism
D000972:Antineoplastic Agents, Phytogenic; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D005047:Etoposide